Sathish, Venkatachalem

Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role for estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. We hypothesize that kisspeptin (Kp)/KISS1R signaling serves this role. Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We explored the role of Kp/KISS1R in regulating ASM proliferation. We report potentially novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10, mitigated PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The antiproliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G 0 /G 1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM proliferation and a potential therapeutic avenue to blunt remodeling in asthma.

AbstractAirway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain‐derived neurotrophic factor (BDNF). The glial cell‐derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+]i. Serum‐deprived ASM cells from non‐asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura‐2 loaded cells, acute GDNF or NRTN alone induced [Ca2+]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1‐Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1‐Fc chelator markedly decreased [Ca2+]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine‐induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Purpose Asthma prevalence has been shown higher in female as compared to male, strongly indicating the involvement of sex steroids in modulating airway hyperreactivity. Airway smooth muscle (ASM) cells are major structural cells of the airway that determine the airway tone. Our recent study demonstrated the presence of estrogen receptors (ERα and ERβ) in ASM cells and that both ER's are significantly upregulated during asthma and/or inflammation with pronounced ERβ expression. We previously reported that estrogen acutely reduces agonist‐induced intracellular calcium ([Ca 2+ ] i ) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca 2+ ] i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca 2+ ] i regulation in ASM cells, with enhanced functionality during inflammation. Methods Human ASM cells were enzymatically dissociated from tissues obtained incidental to lung surgery (Mayo Clinic). Asthmatic and non‐asthmatic ASM cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence or absence of 17β‐estradiol (E 2 ), various ERα (PPT, THC) or ERβ (WAY, FERB, DPN) agonists and antagonists (MPP,PHTPP) for 24h. Cells were incubated with calcium indicator dye Fura‐2 AM and [Ca 2+ ] i responses in these cells were measured using various bronchoconstrictors such as bradykinin (BK,10nM), acetylcholine (Ach,1μM), histamine (His,10μM) and KCl (100μM) under Olympus epifluorescence imaging system. Results In both asthmatic and non‐asthmatic human ASM cells, ERβ agonists reduced [Ca 2+ ] i response to BK, His, Ach as compared to vehicle. Conversely E 2 and ERα agonists showed no change or a slight increase in [Ca 2+ ] i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca 2+ ] i in human ASM cells was further confirmed by ERβ siRNA and ERβ overexpression studies. In the presence of inflammation induced by TNFα or IL‐13, ERβ agonists significantly reduced [Ca 2+ ] i response, while E 2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca 2+ ] i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca 2+ ] i increase, which was comparable to the effect of nifedipine. [Ca 2+ ] i reuptake pump (SERCA) which sequesters the cytosolic calcium into sarcoplasmic reticulum showed increased expression and activity in ASM cells with ERβ activation during inflammation. Conclusion Overall, our data highlights the presence of divergent ER signaling in [Ca 2+ ] i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca 2+ ] i during inflammation, through both L‐type channel inhibition and increased SERCA activity. These varied signaling mechanisms could play a crucial role in smooth muscle contractility and tone in asthma. Support or Funding Information Supported by NIH grants R01 HL123494 (Venkatachalem), and R01 HL088029 (Prakash). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background The pathophysiology of asthma involves inflammation and altered structure and function of airway smooth muscle (ASM). There is emerging evidence that asthma affects adult women disproportionately with more severity than men. Also, our recent findings suggest that differential estrogen receptors (ER), ERα and ERβ expression and function are present in asthmatic ASM cells. Due to high oxygen environment in the lungs and the presence of inflammation, asthmatic ASM cells undergo oxidative stress. Mitochondrial dysfunction due to this oxidative stress plays a crucial role in the bioenergetics pathways involved in asthma. In this study, we hypothesize that differential ER signaling affects mitochondrial dynamics (fission/fusion) in ASM, which subsequently affects reactive oxygen species (ROS) generation and intracellular Ca 2+ ([Ca 2+ ] i ) buffering capacity of mitochondria. Methods Human ASM cells were enzymatically dissociated from lung samples of non‐asthmatics undergoing thoracic surgery at Mayo Clinic. Cells were grown onto 8‐well labtek plates and treated with pro‐inflammatory cytokine, TNFα (20 mg/mL) in the presence or absence of 1 nM 17β‐estradiol (E 2 ) which is a nonselective ER agonist, 10 nM PPT (ERα agonist) or 10 nM WAY (ERβ agonist) for 24h. For morphology studies, the cells were loaded with Mitotracker Green and then imaged on the Bio‐Tek Lionheart FX Live Cell Imager. Form factor and aspect ratio are used as parameters for determining fission vs fusion in mitochondria and these were calculated using MATLAB. Western blot and qPCR techniques were performed to access the protein and gene expression of the fission and fusion proteins Dynamin Related Protein1 (Drp1) and Mitofusion2 (Mfn2). ROS measurement was performed by loading cells with MitoSOX Red and their fluorescence intensity was recorded on the Live Cell Imager. Results In mitotracker loaded ASM cells, ERβ agonist WAY was effective in increasing the form factor and aspect ratio as compared to vehicle. In addition, ERβ was effective in reducing TNFα induced reduction in form factor and aspect ratio. However, both E 2 and ERα agonist, PPT, did not show any changes in form factor and aspect ratio of TNFα exposed ASM cells. Western analysis and gene expression studies showed higher expression of Drp1 and lower expression of Mfn2 in TNFα treated cells. Pre‐treatment with ERβ‐agonist reversed the altered expression of Drp1 and Mfn2 in TNFα treated cells. Using MitoSOX Red, it was observed that ERβ agonist WAY treated cells showed lower ROS generation as compared to TNFα. Conclusion Our data suggests a novel estrogen signaling in ASM cells for maintaining the structure of mitochondria. ERβ is effective in ameliorating the inflammation due to TNFα and hence preserves the morphology of mitochondria during inflammation. Further studies will confirm the subsequent alterations in mitochondrial ROS generation and [Ca 2+ ] i buffering capacity due to this altered mitochondrial morphology. Support or Funding Information Supported by the UGSRF fellowship by APS (Jesme) and NIH grants R01 HL123494, (Venkatachalem), and R01 HL 088029 (Prakash) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Airway inflammation leads to increased intracellular Ca 2+ ([Ca 2+ ] i ) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na + /Ca 2+ exchange (NCX) may contribute to ASM [Ca 2+ ] i regulation. However, the roles of NCX‐mediated Ca 2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca 2+ influx as well as efflux (measured as slopes of changes in [Ca 2+ ] i ) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na + ] i using the fluorescent Na + indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca 2+ ] i or falling [Na + ] i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca 2+ ] i. Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca 2+ stores is an important determinant of [Ca 2+ ] i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca 2+ ] i oscillations representing repetitive SR Ca 2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca 2+ ] i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca 2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca 2+ ] i responses, and slowed or inhibited ongoing [Ca 2+ ] i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca 2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17 ‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca 2+ ] i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca 2+ release and reuptake, likely through PLB. Supported by NIH grant HL74309 (GCS)

There is increasing interest in the role of hydrogen sulfide (H 2 S) as a gasotransmitter similar to nitric oxide and carbon monoxide. In this study, we examined the role of H 2 S in airway smooth muscle (ASM) inflammation. H 2 S is produced by the enzymes cystathionine beta‐synthase (CBS) and cystathionine gamma‐lysase (CTH). Both of these enzymes were detected in human ASM by Western blot analysis. In tissues exposed to the pro‐inflammatory cytokine tumor necrosis factor‐a (TNFα) both CTH and CBS expression were upregulated. In ASM strips contracted with 10 µM ACh, only exposure to 1 mM of the H 2 S donor NaHS induced relaxation. Human ASM cells loaded with fura‐2 demonstrated a dose‐dependent decrease in intracellular calcium concentration ([Ca 2+ ] i ) to NaHS (50‐250 μM), in the presence of either 1 μM ACh or 10 μM histamine. However, sodium sulfide (Na 2 S, 100 µM up to 1 mM) showed no effect on [Ca 2+ ] i . Exposure to 60 mM KCl in the presence of either NaHS or Na 2 S (50 μM‐3 mM) did not affect [Ca 2+ ] i . Relaxant effects of NaHS or Na 2 S on ACh‐induced [Ca 2+ ] i elevation were absent when experiments were conducted in zero extracellular Ca 2+ . These results suggest that H 2 S relaxes ASM and reduces [Ca 2+ ] i by affecting Ca 2+ influx, potentially via voltage‐gated channels, but does not affect sarcoplasmic reticulum Ca 2+ release. The relevance of endogenous H 2 S generation in ASM, and its role in infalmmation needs to be explored further. Supported by the Flight Attendants Medical Research Institute (FAMRI), NIH grant HL74309, and the Department of Anesthesiology, Mayo Clinic.

Intracellular Ca 2+ ([Ca 2+ ] i ) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are key components of ASM [Ca 2+ ] i regulation. Ca 2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca 2+ ] i . We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca 2+ ] i responses to ACh or bradykinin (zero extracellular Ca 2+ ) and found significant reduction in rates of fall of [Ca 2+ ] i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca 2+ ] i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function. Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.

Purpose Bronchial asthma is a chronic inflammation of the airways that is characterized by airway remodeling, hyperactive responsiveness and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest that there is a higher incidence of asthma in pre‐menopausal women and aging men, strongly indicating the role of sex steroids in modulating the airway tone. A major sex steroid, estrogen mediates its role through estrogen receptors (ER) ‐ ERα and ERβ. Our recent study demonstrated that these receptors are expressed in both male and female ASM cells and their expression is upregulated in the presence of inflammation/asthma. However, the role of ER signaling in asthma or genomic mechanisms in intracellular Ca 2+ ([Ca 2+ ] i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca 2+ ] i in human ASM cells. Methods Human ASM cells were enzymatically dissociated from tissue obtained incidental to lung surgery from Mayo Clinic (IRB approved). Cells were plated onto 8‐well Lab‐Tek plates and grown to 70% confluence in a 5% CO 2 humidified incubator. Cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence and absence of 1nM 17β‐estradiol (E 2 ), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24h. Cells were washed with HBSS and incubated with 3 μm Fluo‐3 for 1h for calcium imaging. The [Ca 2+ ] i levels are measured from the intensity change observed following exposure to 10μM histamine under both physiological (2 mM) and zero extracellular calcium using Biotek LFX Fluorescence Imager. Results In fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E 2 ) was effective in reducing [Ca 2+ ] i levels. TNFα and IL‐13 significantly increased [Ca 2+ ] i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca 2+ ] i in ASM cells, while PPT did not draw out any notable changes compared to TNFα or IL‐13 treatment alone. Addition of nifedipine (1μM, L‐type calcium channel blocker) resulted in a ~40% decrease in [Ca 2+ ] i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca 2+ ] i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca 2+ reuptake, extracellular Ca 2+ was removed by perfusion with zero Ca 2+ HBSS, and Ca 2+ influx was non‐specifically blocked with 1 mM LaCl 3. Under these conditions, the [Ca 2+ ] i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca 2+ ] i sequestration to SR (SERCA activity). In both control ASM cells as well as in those exposed to cytokines, WAY increased the rate of fall of [Ca 2+ ] i responses to histamine, suggesting greater sequestration of [Ca 2+ ] i. Conclusions Overall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca 2+ ] i in the inflamed ASM via receptor‐mediated genomic action. Specifically, ERβ activation is more effective in the presence of inflammation. Support or Funding Information Supported by NIH grants R01 HL123494 and R01 HL123494‐02S1 (Venkatachalem) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .